There is widespread concern over potential adverse human health effects associated with exposures to endocrine-active industrial chemicals, and regulatory agencies will be undertaking large scale screening and testing for estrogen, androgen and thyroid hormone receptor agonists/antagonist activities. Standardized screening and testing for industrial-derived estrogenic (xenoestrogens) will provide assay-specific potency data but may not be predictive for their tissue-specific estrogen receptor (ER) agonist or antagonist activities. Based on results of preliminary studies with bisphenol A, we hypothesize that different structural classes of xenoestrogens and naturally-occurring estrogenic compounds exhibit "unique" estrogenic activities and unique biology that cannot be determined using traditional bioassays. Therefore, Objective 1 of this proposal will be the following. 1. Six different structural classes of estrogenic compounds will be investigated in the HepG2 cell assay to determine their specific mechanism-based ER action. 2. Ligand structure-dependent differences will also be determined in a new androgen-dependent assay that distinguishes between D2 and diethylstilbestrol (DES) and may be useful for other xenoestrogens. 3. Ligand-dependent differences in activation of ERalpha vs. ERbeta will also be determined using new assays developed in this laboratory that involve gene activation through GC-rich Sp1 binding sites. Aim 2 is also focused on improving mechanism based hazard/risk assessment by determining ER-subtype specific induction of gene expression. We hypothesize that ERalpha and ERbeta induce overlapping and different genes and the proposed studies will use a new specific inhibitor of ERbeta action coupled with a suppression subtractive hybridization (SSH) technique to identify ER-subtype-specific gene expression. Objectives 1 and 2 will give new mechanism-based data on xenoestrogen-mediated ER action and provide a more rational basis for risk/hazard assessment of these compounds.